ORIGINAL ARTICLE

Effect of resveratrol on cartilage protection and apoptosisinhibition in experimental osteoarthritis of rabbit

Jing Wang • Jie-Sheng Gao • Jin-Wei Chen •

Fen Li • Jing Tian

Received: 10 June 2010 / Accepted: 30 December 2010 / Published online: 15 February 2011

� Springer-Verlag 2011

Abstract To observe the effect of resveratol on carti-

lage,chondrocyte apoptosis, and nitric oxide in experi-

mental osteoarthritis (OA) of rabbit and to study the

mechanism of resveratol in the treatment of osteoarthritis.

Thirty New Zealand rabbits were randomly divided into 5

groups: group A (normal control group), group B (model

control group), group C (resveratrol intervention high-

dosage group), group D (resveratrol intervention middle

dosage group), and group E (resveratrol intervention

low-dosage group). The model of OA of the knee was

established using Hulth technique in groups B, C, D, and E.

After 4 weeks, group A and group B rabbits were admin-

istered daily a knees injection of dimethylsulfoxide

(DMSO), whereas groups C, D, and E were administered

daily a knees injection of resveratrol in DMSO in different

dosages for 2 weeks. Daily dosage for rabbits of groups C,

D, and E was fixed at 50, 20, and 10 lmol/kg, respectively.

Then, the rabbits were killed, and the lateral cartilage

sections of right femoral medial condyle were evaluated

using a histological scoring system (H&E and safranin-O

staining) and analyzed by TUNEL for apoptosis. Nitric

oxide (NO) in synovial fluid was measured by nitrate

reduction method. Histological evaluation of cartilage tis-

sue revealed a significantly reduced cartilage destruction;

the evaluation also revealed the loss of matrix proteoglycan

content in cartilage in resveratrol intervention groups

compared to the model control. Resveratrol reduced the

apoptosis rate of chondrocyte and level of NO in the

synovial fluid. In the above experiments of OA rabbits,

the protective effects of resveratrol were enhanced with

increased resveratrol dosage. Resveratrol controls the

progression of experimental OA. One of the mecha-

nism(s) responsible for this effect would include lowering

of the apoptosis rate of chondrocyte and reducing the

production of NO in experimental OA.

Keywords Resveratrol � Cartilage �Osteoarthritis � Apoptosis � Chondrocyte

Introduction

Osteoarthritis (OA) is a chronic disease characterized by

the progressive degeneration of cartilage, causing pain, and

loss of articular function. The primary pathogenic events in

OA include loss and abnormal remodeling of cartilage

extracellular matrix (ECM). Chondrocytes are the only

type of cells constituting the articular cartilage. Their

functions include maintaining tissue homeostasis,

responding to injury, and executing the cartilage remod-

eling process that characterizes OA. The earlier concepts

on OA pathogenesis merely focused on the role of syn-

thesis and degradation of the ECM in the articular carti-

lage. More recent findings indicate that chondrocyte death

and survival are closely linked with cartilage matrix

integrity [1]. Chondrocytes are differentiated from mes-

enchymal cells during embryonic development. Differen-

tiated chondrocytes synthesize sufficient amounts of

cartilage-specific ECM—including type II collagen and

proteoglycan—to maintain matrix integrity [1, 2]. It has

been found that this homeostasis is destroyed by chon-

drocyte apoptosis in the progressive stage of OA. So,

apoptosis inhibition appears feasible and should be con-

sidered and explored as a therapeutic target of OA.

Researches showed that there appears to be at least two

J. Wang � J.-S. Gao (&) � J.-W. Chen � F. Li � J. Tian

Department of Rheumatology, The Second Xiangya Hospital of

Central-South University, Changsha 410011, Hunan, China

e-mail: jingmail000@126.com

123

Rheumatol Int (2012) 32:1541–1548

DOI 10.1007/s00296-010-1720-y

independent pathways for the induction of chondrocyte

apoptosis—the Fas and the NO pathways [3]. Pro-inflam-

matory cytokines tumor necrosis factor-a (TNF-a) and

interleukin-1b (IL-1b), which are thought to play an

important role in the pathogenesis of arthritis, differentially

regulate the apoptotic pathway in human chondrocytes by

NO pathway [4]. In addition, NO mediates the inflamma-

tory response involved in the matrix metalloproteinases

degradation, thereby inhibiting the synthesis of collagen

and proteoglycans [5].

Resveratrol (trans-3, 4-trihydroxystilbene) is a natural

phytoalexin (polyphenolic compound) found in the skin of

red grapes, cranberries, peanuts, and root extracts of the

weed Polygonum cuspidatum. It has been reported that

resveratrol has antitumor activity, immunomodulatory,

antioxidative, anti-inflammatory functions, apart from

numerous biological activities [6].

In this study, we intend to reveal the effect of resveratol

on cartilage, chondrocyte apoptosis, and nitric oxide in

experimental osteoarthritis (OA) of rabbit.

Materials and methods

Animals and surgical model

In this study, all experiments were performed in accor-

dance with the guidelines for animal research from the

National Institutes of Health and were approved by the

Committee on Animal Research at the Second Xiangya

Hospital.

Resveratrol was purchased from Sigma (500 mg) and

prepared according to the manufacturer’s protocol. In brief,

the outline of the procedure is as follows: 100 lmol res-

veratrol was dissolved in dimethylsulfoxide (DMSO) and

prepared stock resveratrol solution. It was diluted in serum

physiologic as 30 lMol in 100 lL.

Thirty adult New Zealand white rabbits (equal number

of male and female rabbits, weight 1.5–2.5 kg) were

divided randomly into 5 groups (each group consists of

6 rabbits): group A (normal control group), group B (model

control group), group C (resveratrol intervention high-

dosage group), group D (resveratrol intervention mid-

dle dosage group), and group E (resveratrol intervention

low-dosage group).

The model of OA was established in groups B, C, D, and

E using the Hulth-Telhag modeling method. The surgical

procedure for the modeling groups was performed under

sterile conditions in an animal surgical suite under general

endotracheal anesthesia (Halotane 2%). Intramuscular

antibiotic (Enrofloxacin 5 mg/kg) was administered. A

midline skin incision was then made over the right knee: a

medial parapatellar incision was made through the

retinaculum. The medial collateral ligament was sharply

divided.

A medial parapatellar arthrotomy was then performed,

and the patella was dislocated laterally. Care was taken

to protect and retract the vascular intraarticular fat pad.

With the knee flexed, the anterior cruciate ligament

and the posterior cruciate ligament were transected. The

knee joint was then dislocated to excise the medial

meniscus.

The joint was irrigated with sterile saline solution. The

capsule and the synovium were then closed together using

a 4.0 interrupted Vicryl and the skin was closed. A sham

procedure was performed on the left hind limb serving as a

control within the same animal. An arthrotomy was per-

formed, as described above. After dislocating the patella

laterally, the knee was irrigated; but the ligaments and

menisci were left intact. Postoperatively, the rabbits were

kept in single-subject cages for 4 days and analgesics

(Buprenex) were administered as per requirement. The

rabbits were allowed to bear weight as per their individual

tolerance.

After 4 days, the rabbits were transferred to three large

free-roaming communal cages (10 9 15 feet) where they

had unlimited activity. Four weeks later, rabbits of group A

and group B were administered daily knees injection con-

taining DMSO, while rabbits of groups C, D, and E were

administered daily knees injection containing resveratrol in

DMSO at different dosages for 2 weeks. Daily dosage of

groups C, D, and E were 50, 20, and 10 lmol/kg, respec-

tively. Then, the rabbits were killed, and the lateral carti-

lage sections of right femoral medial condyle and synovial

fluid were analyzed.

Histology

For histologic examination, the cartilage specimens of

knees were fixed in 10% neutral-buffered formalin; then

they were decalcified with 10% Na2 ethylenediaminete-

traacetate (EDTA) buffered at pH 7.4 and embedded in

paraffin. Several sections of 4 mm thickness were cut from

the lateral articular cartilage of right femoral medial con-

dyle on a rotary microtome.

For histological evaluation, sagittal sections derived

from chondral tissues were stained with hematoxylin and

eosin (H&E) and safranin O. A semiquantitave scoring on a

grading scale for cartilage erosions was used by an inde-

pendent observer for each knee. A characteristic parameter

in arthritis is the progressive loss of articular cartilage. The

depth of cartilage erosion determined by H&E staining was

graded according to Mankin scoring criteria (total score: 14

scores). In addition, cartilage proteoglycan depletion was

determined using safranin-O staining. The loss of proteo-

glycans was scored from 0 to 3, ranging from fully stained

1542 Rheumatol Int (2012) 32:1541–1548

123

cartilage to destained cartilage or complete loss of articular

cartilage.

TUNEL method

The DNA cleavage was assessed by the terminal deoxy-

nucleotidyl transferase–mediated dUTP nick end labeling

(TUNEL); therefore, the ‘‘In situ cell death detection,

fluorescein’’ kit was used. Cells were gently washed with

phosphate-buffered saline (PBS) and fixed with 250 ll of

4% (w/v) paraformaldehyde prepared freshly in PBS

(pH = 7.4) for 30 min at room temperature.

After washing twice in PBS, cells were incubated in

200 ll of permeabilization solution (0.1% [w/v] Triton

X-100 in 0.1% [w/v]sodium citrate) for 2 min on ice. Cells

were again washed in PBS before incubation with 55 ll of

TUNEL reagents (terminal deoxynucleotide transferase

and fluorescein-labeled nucleotide mixture) or 55 ll of

LABEL (without terminal transferase) used as negative

control for 2 h at 37�C in a humidified chamber.

As a negative control, staining was performed without

terminal deoxynucleotidyl transferase. The apoptosis ratio

(%) was measured: it is defined as ratio between the total

number of positive nuclei and the total number of the

present cells in the six randomly allocated cellular fields.

Positive nuclei stained brown and negative ones stained

blue.

Nitric oxide assays

NO was measured in terms of NO metabolite (nitrates and

nitrites) release using a Nitric Oxide Detection Kit

(Shenzhen Jingmei Bioengineering Institute, China),

according to the manufacturer’s instructions. NO is

chemically active and rapidly converts to NO3- and NO2-

in vivo. This method uses nitrate reductase to specifically

reduce NO3- to NO2-, and the content of NO2- is deter-

mined colorimetrically. This kit is sensitive, stable, and

simple to use, having a major advantage of measuring the

total amount of NO3- and NO2- through nitrate reductase.

Statistical analysis of data

The data obtained in this study were introduced into a

database specifically designed for analysis with SPSS 11 .0

statistical package. Data were expressed as the mean ± SD

for each group. One-way analysis of variance (ANOVA)

with Tukey’s multiple comparison test was used to com-

pare differences in nitric oxide production and apoptosis

rates among the groups. In all cases, P \ 0.05 was con-

sidered as significant.

Results

All rabbits in each experimental group completed the

study. No signs of resveratrol or DMSO toxicity were

noted. The levels of daily activity were similar in all

rabbits.

Histological analyses

In groups B and A, significantly increased cartilage

destruction was determined by H&E staining (1.1 ± 0.32

vs 9.2 ± 0.57 and P \ 0.05). Cartilage destruction in

groups C, D, and E (4.2 ± 0.78, 6.9 ± 0.36, 7.8 ± 0.41)

was less compared to that in group B. According to Mankin

scoring criteria, significant differences of the scores were

found among groups C, D, and E (P \ 0.05) (Table 1;

Fig. 1).

In addition, loss of matrix proteoglycan content in the

cartilage was much lower in group A compared to group B

(0.4 ± 0.12 vs 2.6 ± 0.34 and P \ 0.05), as determined by

safranin-O staining. Specimens of cartilage from the group

B exhibited morphologic changes characteristic of arthritis.

These included fibrillation and fissures of the cartilage

surface and loss of safranin-O staining. Groups C, D, and E

(1.3 ± 0.23, 1.9 ± 0.12, 2.1 ± 0.27) reduced the loss of

matrix proteoglycan content compared to group B

(P \ 0.05) (Table 1; Fig. 2).

Apoptosis by tunnel

The apoptosis rates of chondrocyte in groups B, C, D, and

E were significantly higher than that of group A (31.64 ±

4.05%, 18.15 ± 2.21%, 22.92 ± 3.57%, 27.42 ± 2.71%,

3.28 ± 1.02%, P \ 0.01). The apoptosis rates of chon-

drocyte in groups C, D, and E were decreased com-

pared with that of group B (P \ 0.01, P \ 0.01, and

P \ 0.05, respectively). Significant differences of apopto-

sis rate were found among groups C, D, and E (P \ 0.05)

(Fig. 3).

Nitric oxide assays

The nitric oxide content of synovial fluid in groups B, C, D,

and E (138.71 ± 4.96; 87.13 ± 2.87, 93.36 ± 2.63,

105.42 ± 3.85) were significantly higher than that of group

A (46.39 ± 5.98 and P \ 0.05). The nitric oxide content of

synovial fluid in groups C, D, and E were decreased

compared with that of group B (P \ 0.05, respectively).

Significant differences of nitric oxide content were found

among groups C, D, and E (P \ 0.05).

Rheumatol Int (2012) 32:1541–1548 1543

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Table 1 Histological analyses by H&E staining and safranin-O staining with a grading scale, apoptosis rates, and nitric oxide content of

synovial fluid in each group

Groups Dosage of resveratrol

(lmol/kg/day)

Cartilage destruction

by H&E (scores)

Proteoglycan loss by

safranin-O staining (scores)

Apoptosis rates of

chondrocyte (%)

Nitric oxide content of

synovial fluid (lmol/L)

Group A – 1.1 ± 0.32 0.4 ± 0.12 3.28 ± 1.02 46.39 ± 5.98

Group B – 9.2 ± 0.57 2.6 ± 0.34 31.64 ± 4.05 138.71 ± 4.96

Group C 50 4.2 ± 0.78 1.3 ± 0.23 18.15 ± 2.21 87.13 ± 2.87

Group D 20 6.9 ± 0.36 1.9 ± 0.12 22.92 ± 3.57 93.36 ± 2.63

Group E 10 7.8 ± 0.41 2.1 ± 0.27 27.42 ± 2.71 105.42 ± 3.85

Fig. 1 Representative

histologic staining (hematoxylin

and eosin, H&E) of lateral

cartilage sections in all groups.

H&E staining showed

resveratrol at different dosage

prevented cartilage destruction

of OA, including cartilage

thinning, hypocellularity, and

fissures of the cartilage surface

to different extents

1544 Rheumatol Int (2012) 32:1541–1548

123

Discussion

Resveratrol (3,5,40-trihydroxystilbene) is a phytoalexin

present in a wide variety of plant species, including mul-

berries, peanut sand grapes, and thus is a constituent of the

human diet. This compound, like other members of stilbene

family, is produced in response to pathogen attack, UV-

irradiation, and exposure to ozone. Researches showed that

resveratrol have anti-inflammatory, anticarcinogen, anti-

aging, and antioxidant properties [6]. Resveratrol attenu-

ated oxidative stress and inhibited iNOS expression,

reducing the production of NO in many kinds of cell in

vivo and in vitro [7].

Apart from the observations, resveratrol has been shown

to be a potent inhibitor of both NF-kB activation and NF-

kB-dependent gene expression [8, 9]. Resveratrol inhibits

COX-2 and iNOS expression by blocking NF-kB activation

[10]; it also blocks TNF-a and IL-1b-induced activation of

the NF-kB [11]. NF-kB can regulate chondrocyte apoptosis

induced by NO, IL-1b, IL-17 [12–16].

In vitro study, Shakibaei and his colleagues found the

antiapoptotic effects of resveratrol on IL-1b-stimulated

Fig. 2 Representative

histologic staining with safranin

O of lateral cartilage sections in

all groups. Resveratrol at

different dosage reduced loss of

matrix proteoglycan content in

the cartilage by safranin-O

staining

Rheumatol Int (2012) 32:1541–1548 1545

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chondrocytes [17]. Moreover, resveratrol was defensive to

the IL-1b-induced catabolic effects of chondrocytes by

elevating proteoglycan synthesis, suppressing COX–2

expression and enzyme activity, and decreasing production

of MMPs 1, 3, and 13 [18]. A vivo study demonstrated that

intraarticular injections of resveratrol reduced the severity

of cartilage lesions by histologic examination in the

experimental OA model [19].

In this study, we also found that resveratrol exhibited

cartilage protective effect in the experimental OA model of

rabbits. For histologic examination, the degree of the car-

tilage lesions and loss of matrix proteoglycan content in the

cartilage were significantly improved by treatment with

resveratrol.

Apoptosis plays a pivotal role in OA pathogenesis. The

apoptosis ratio is higher (18–51%) in the articular cartilage of

OA than that of the normal [1, 2, 20]. Our result

(31.64 ± 4.05%) is in accordance with these studies. The

number of apoptotic cells was correlated with matrix degra-

dation, the corruption of fibrillar architecture, and the OA

grade [1, 2]. In the animal experimental study, intraarticular

administration of the pan-caspase inhibitor Z-VAD-FMK,

which can inhibit apoptosis significantly, reduced cartilage

degradation in experimental OA [21]. This result provides

Fig. 3 Specimen of cartilage

tissue from group A showed few

apoptotic chondrocytes. A few

apoptotic chondrocytes defused

distributed in full-thickness

cartilage, especially in middle

and surface layer in group B.

Resveratrol reduced the

apoptosis rates of chondrocyte

in OA

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direct support for a role of apoptosis in OA pathogenesis,

suggesting that inhibition of chondrocyte apoptosis would be

an effective therapeutic approach for OA. A study showed

antiapoptotic effects of resveratrol on IL-1b-stimulated

chondrocytes as an in vitro model of OA [18]. In the animal

model of OA, our study presented resveratrol decrease

apoptosis rates of chondrocyte. The results demonstrated

resveratrol inhibit chondrocyte apoptosis of OA in vivo. It also

implied inhibition of chondrocyte apoptosis may be one of

the mechanisms resveratrol protecting cartilage destruction.

NO plays an important role in synthesis, degradation of

ECM, and chondrocyte apoptosis. A study in an experi-

mental model of OA revealed a selective inhibition of

iNOS by L-NIL and the subsequent decreased production

of NO resulting in decreased level of chondrocyte apop-

tosis and the progression of experimental OA [22, 23]. Our

study shown that resveratrol reduce the production of NO

in the synovial fluid. The results indicated that resveratrol

reduced the production of NO and interfered in the NO

pathway for the induction of chondrocyte apoptosis. The

protective effects of resveratrol in experimental OA are

attributed, in part, to its capacity to inhibit chondrocyte

apoptosis and reduce the production of NO.

Previous data showed that resveratrol is effective at

concentrations ranging from 5 to 100 lM in chondrocytes in

vitro [17, 18]. Elmali N selected 10 lmol/kg/day resveratrol

as therapeutic dosage in animal model exhibiting protective

effect [19]. In our study, we chose 50, 20, 10 lmol/kg,

respectively, as daily dosages of three groups to identify the

protective effect of resveratrol at different dosages.

Significant differences of Mankin scores, chondrocyte

apoptosis rates, and NO content were found among the

three groups. The protective effects of resveratrol were

enhanced with increased resveratrol dosage. The results

suggested the protective effects of resveratrol were dose

dependent in the dose range of 10–50 lmol/kg.

In conclusion, we have shown that 2 weeks intraartic-

ular administration of resveratrol (10–50 lmol/kg/day)

protect against cartilage destruction by inhibiting chon-

drocyte apoptosis and reducing the production of NO in the

rabbit OA model. From the results of this study, we believe

resveratrol might provide a novel and alternative approach

as a disease-modifying OA drug. We also hope that the

results of this study could bring more studies to confirm the

therapeutic benefits and explore the mechanisms of resve-

ratrol in the treatment of OA.

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